Patent application title: NGF APTAMER AND APPLICATION THEREOF

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Abstract:

The present invention provides an aptamer binding to NGF and capable of
forming a potential secondary structure represented by the formula (I):
##STR00001##
wherein N is one nucleotide selected from the group consisting of A, G,
C, U and T, N11-N13, N21-N23, N32-N38 and N42-N48 are the same or
different and each is a bond or 1 or 2 nucleotides selected from the
group consisting of A, G, C, U and T, N14, N24, N31, N41, N39 and N49 are
the same or different and each is one nucleotide selected from the group
consisting of A, G, C, U and T, N14 and N24, N31 and N41, and N39 and N49
each form a Watson-Crick base pair, N11-N12-N13-N14 and N21-N22-N23-N24
are nucleotide sequences capable of forming a stem structure in
combination, and N31-N32-N33-N34-N35-N36-N37-N38-N39 and
N41-N42-N43-N44-N45-N46-N47-N48-N49 are nucleotide sequences capable of
forming a stem structure in combination.

Claims:

1. An aptamer binding to NGF and capable of forming a potential secondary
structure represented by the formula (I): ##STR00006## wherein N is one
nucleotide selected from the group consisting of A, G, C, U and T,
N11-N13, N21-N23, N32-N38 and N42-N48 are the same or different and each
is a bond or 1 or 2 nucleotides selected from the group consisting of A,
G, C, U and T, N14, N24, N31, N41, N39 and N49 are the same or different
and each is one nucleotide selected from the group consisting of A, G, C,
U and T, N14 and N24, N31 and N41, and N39 and N49 each form a
Watson-Crick base pair, N11-N12-N13-N14 and N21-N22-N23-N24 are
nucleotide sequences capable of forming a stem structure in combination,
and N31-N32-N33-N34-N35-N36-N37-N38-N39 and
N41-N42-N43-N44-N45-N46-N47-N48-N49 are nucleotide sequences capable of
forming a stem structure in combination.

2. The aptamer according to claim 1, wherein N11-N13, N21-N23, N32-N38
and N42-N48 are the same or different and each is one nucleotide selected
from the group consisting of A, G, C, U and T.

3. The aptamer according to claim 1, wherein N14 is U, N24 is A, N31 is
G, N41 is C, N39 is G, and N49 is C.

4. The aptamer according to claim 1, wherein not less than 4 Watson-Crick
base pairs are formed between N32-N33-N34-N35-N36-N37-N38 and
N42-N43-N44-N45-N46-N47-N48.

5. The aptamer according to claim 1, which is the following (a) or (b):
(a) a nucleic acid consisting of a nucleotide sequence selected from SEQ
ID NO: 3, SEQ ID NOs: 9-13, SEQ ID NOs: 22-117 and SEQ ID NOs: 152-168
(wherein uracil may be thymine); (b) a nucleic acid binding to NGF and
consisting of the nucleotide sequence of the above-mentioned (a), wherein
1 to several nucleotides are substituted, deleted, inserted or added.

6. The aptamer according to claim 1, which has a base length of not more
than 50.

7. The aptamer according to claim 1, wherein at least one nucleotide is
modified.

8. The aptamer according to claim 7, which is modified with inverted dT
or polyethylene glycol.

9. The aptamer according to claim 8, wherein the inverted dT or
polyethylene glycol is bound to the 5' end or 3' end of the aptamer.

10. The aptamer according to claim 7, wherein the hydroxyl groups at the
2'-position of a ribose of respective pyrimidine nucleotides are the same
or different and unreplaced or replaced by an atom or group selected from
the group consisting of a hydrogen atom, a fluorine atom and a methoxy
group.

11. The aptamer according to claim 7, wherein the hydroxyl groups at the
2'-position of a ribose of respective purine nucleotides are the same or
different and unreplaced or replaced by an atom or group selected from
the group consisting of a hydrogen atom, a fluorine atom and a methoxy
group.

13. A pharmaceutical composition comprising the aptamer according to
claim 1.

14. An anti-pain agent comprising the aptamer according to claim 1.

15. An anti-inflammatory agent comprising the aptamer according to claim
1.

16. A method of treating or preventing a disease accompanying a pain or
inflammation, comprising administering the aptamer according to claim 1
to a subject in need thereof.

17. (canceled)

Description:

TECHNICAL FIELD

[0001] The present invention relates to an aptamer for NGF, and use
thereof.

BACKGROUND ART

[0002] Nerve growth factor (NGF) is the first neurotrophin identified in
1951, and is an important secretory protein involved in the development
and survival of peripheral and central neurons. It consists of 118 amino
acids, has a molecular weight of 13 kDa, and has S--S bonds at 3
positions in a molecule.

[0003] As NGF receptors, tyrosine kinase-type receptor TrkA with high
affinity and p75 with low affinity which belongs to a tumor necrosis
factor receptor superfamily are known. These receptors act as a homodimer
or heterodimer and are deeply involved in the development and maintenance
of the nervous system. TrkA is a single-pass transmembrane receptor and
has a tyrosine kinase structure in the intracellular domain. When NGF is
bound, tyrosine phosphorylation occurs, the signal is transmitted to the
downstream, and promotion of differentiation and survival maintenance of
the cell occur.

[0004] As family receptors of TrkA, TrkB and TrkC are known. TrkB is bound
to BDNF and NT-4/5, and TrkC is bound to NT-3. p75 shows lower ligand
specificity as compared to TrkA and is also bound to BDNF, NT-3 and
NT-4/5 besides NGF. While p75 is a single-pass transmembrane receptor, it
does not have a tyrosine kinase domain on the cytoplasmic side. Like
TrkA, it is expressed not only in nerve cells but also in non-nerve
cells. This receptor is known to be involved in the promotion of
differentiation and survival maintenance of the cell, as well as related
to the induction of apoptosis and cell migration. The results of crystal
structure analysis have suggested that an NGF homodimer binds to TrkA at
2:2 and to p75 at 2:1. An NGF homodimer sometimes binds to a heterodimer
of TrkA and p75.

[0005] It is well known that NGF plays a key role in the nervous system.
It has been clarified that NGF has an action to maintain survival of
cholinergic neuron and is considered to be related in some way to
Alzheimer's disease. In addition, since intracerebral administration of
NGF improves memory disorders of old rats, it is also expected as a
therapeutic drug for senile dementia.

[0006] NGF is also related to inflammation, and increased expression of
NGF has been observed in patients with inflammatory diseases and
inflammatory animal models. Systemic lupus erythematosus, multiple
sclerosis, psoriasis, arthritis, interstitial cystitis, asthma and the
like are the examples thereof. It has been reported that the synovial
fluid of patients with rheumatoid arthritis shows higher NGF
concentration. In addition, increased NGF expression in rheumatoid
arthritis model rats, and increase in mast cells and increased NGF
expression in arthritis model mouse have been reported.

[0007] NGF is deeply involved in pain. When NGF is subcutaneously
administered to human, a deep pain such as muscular pain continues for
several days, and hyperalgesia of the injection site occurs. NGF knockout
mouse and TrkA knockout mouse lacks unmyelinated nerve and do not feel
pain. When NGF is intraperitoneally administered at 1 mg/kg to a mature
rat, hyperalgesia against noxious heat and mechanical stimuli occurs. NGF
transgenic mouse shows hyperalgesia unaccompanied by inflammatory
conditions. In addition, it is known that the TrkA gene of patients with
congenital insensitivity to pain with anhidrosis (CIPA) has abnormality,
and pain sensation decreases when NGF gene has abnormality.

[0008] From the above, it is appreciated that an NGF inhibitor can be used
as a therapeutic drug for pain such as nociceptive pain, inflammatory
pain, neuropathic pain, carcinomatous pain, fibromyalgia pain and the
like.

[0009] In recent years, applications of RNA aptamers to medicaments,
diagnostic agents, and test drugs have been drawing attention; some RNA
aptamers have already been in clinical study stage or in practical use.
In December 2004, the world's first RNA aptamer drug, Macugen, was
approved as a therapeutic drug for age-related macular degeneration in
the US. An RNA aptamer refers to an RNA that binds specifically to a
target substance such as a protein, and can be prepared using the SELEX
(Systematic Evolution of Ligands by Exponential Enrichment) method
(Patent references 1-3). In the SELEX, an RNA that binds specifically to
a target substance is selected from an RNA pool with about 1014
different nucleotide sequences. The RNA structure used has a random
sequence of about 40 residues, which is flanked by primer sequences. This
RNA pool is allowed to be assembled with a target substance, and only the
RNA that has bound to the target substance is collected using a filter
and the like. The RNA collected is amplified by RT-PCR, and this is used
as a template for the next round. By repeating this operation about 10
times, an RNA aptamer that binds specifically to the target substance can
be acquired.

[0010] Aptamer drugs, like antibody drugs, can target extracellular
factors. With reference to many scientific papers and other reference
materials in the public domain, aptamer drugs are judged to potentially
surpass antibody drugs in some aspects. For example, aptamers often show
higher binding force and higher specificity than do antibodies. Aptamers
are unlikely to undergo immune elimination, and adverse reactions
characteristic of antibodies, such as antibody-dependent cell-mediated
cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), do not
occur with the use of aptamers. From the aspect of delivery, since
aptamers are about 1/10 of antibody in size, delivery of a drug to the
object site is easier. Since aptamers are produced by chemical synthesis,
various modifications can be made easily, reduction of cost by
large-scale production is possible. Meanwhile, the blood half-lives of
aptamers are generally shorter than those of antibodies; however, this
property is sometimes advantageous in view of toxicity. These facts lead
to the conclusion that even when the same molecule is targeted, aptamer
drugs potentially surpass antibody drugs.

[0011] The present inventors have produced an aptamer which binds to NGF
and inhibits binding of NGF and an NGF receptor, and found that the
aptamer inhibits a neurite outgrowth activity of NGF (patent document 4).
Patent document 5 describes an aptamer to NGF, which is obtained by
automated SELEX, and patent document 6 describes an altered product and a
modified product of the aptamer obtained in patent document 4.

DOCUMENT LIST

Patent Documents

[0012] patent document 1: WO91/19813

[0013] patent document 2:
WO94/08050

[0014] patent document 3: WO95/07364

[0015] patent document 4:
WO2010/035725

[0016] patent document 5: WO02/077262

[0017] patent
document 6: WO03/070984

SUMMARY OF THE INVENTION

Problems to be Solved by the Invention

[0018] The present invention aims to provide an aptamer for NGF, a method
of utilizing the same, and the like. Particularly, the present invention
aims to provide an NGF aptamer suitable for use as a pharmaceutical
product, namely, an aptamer having a short chain length, a high NGF
activity (neurite outgrowth activity, TF-1 cell proliferation activity)
inhibitory activity, and high specificity for NGF.

Means of Solving the Problems

[0019] The present inventors have conducted intensive studies in an
attempt to solve the aforementioned problems and succeeded in producing a
higher quality NGF aptamer showing an IC50 value of 1 nM or below as
to neurite outgrowth inhibition, and a remarkably high neurite outgrowth
inhibitory activity as compared to conventionally-known NGF aptamers,
which resulted in the completion of the present invention.

[0020] Accordingly, the present invention provides the following.

[1] An aptamer binding to NGF and capable of forming a potential
secondary structure represented by the formula (I):

##STR00002##

wherein N is one nucleotide selected from the group consisting of A, G,
C, U and T,

[0021] N11-N13, N21-N23, N32-N38 and N42-N48 are the same or different and
each is a bond or 1 or 2 nucleotides selected from the group consisting
of A, G, C, U and T,

[0022] N14, N24, N31, N41, N39 and N49 are the same or different and each
is one nucleotide selected from the group consisting of A, G, C, U and T,

[0023] N14 and N24, N31 and N41, and N39 and N49 each form a Watson-Crick
base pair,

[0024] N11-N12-N13-N14 and N21-N22-N23-N24 are nucleotide sequences
capable of forming a stem structure in combination, and

[0025] N31-N32-N33-N34-N35-N36-N37-N38-N39 and
N41-N42-N43-N44-N45-N46-N47-N48-N49 are nucleotide sequences capable of
forming a stem structure in combination.

[2] The aptamer according to the above-mentioned [1], wherein N11-N13,
N21-N23, N32-N38 and N42-N48 are the same or different and each is one
nucleotide selected from the group consisting of A, G, C, U and T. [3]
The aptamer according to the above-mentioned [1] or [2], wherein N14 is
U, N24 is A, N31 is G, N41 is C, N39 is G, and N49 is C. [4] The aptamer
according to any of the above-mentioned [1] to [3], wherein not less than
4 Watson-Crick base pairs are formed between N32-N33-N34-N35-N36-N37-N38
and N42-N43-N44-N45-N46-N47-N48. [5] The aptamer according to the
above-mentioned [1], which is the following (a) or (b): (a) a nucleic
acid consisting of a nucleotide sequence selected from SEQ ID NO: 3, SEQ
ID NOs: 9-13, SEQ ID NOs: 22-117 and SEQ ID NOs: 152-168 (wherein uracil
may be thymine); (b) a nucleic acid binding to NGF and consisting of the
nucleotide sequence of the above-mentioned (a), wherein 1 to several
nucleotides are substituted, deleted, inserted or added. [6] The aptamer
according to any of the above-mentioned [1] to [5], which has a base
length of not more than 50. [7] The aptamer according to any of the
above-mentioned [1] to [6], wherein at least one nucleotide is modified.
[8] The aptamer according to the above-mentioned [7], which is modified
with inverted dT or polyethylene glycol. [9] The aptamer according to the
above-mentioned [8], wherein the inverted dT or polyethylene glycol is
bound to the 5' end or 3' end of the aptamer. [10] The aptamer according
to any of the above mentioned [7] to [9], wherein the hydroxyl groups at
the 2'-position of a ribose of respective pyrimidine nucleotides are the
same or different and unreplaced or replaced by an atom or group selected
from the group consisting of a hydrogen atom, a fluorine atom and a
methoxy group. [11] The aptamer according to any of the above-mentioned
[7] to [9], wherein the hydroxyl groups at the 2'-position of a ribose of
respective purine nucleotides are the same or different and unreplaced or
replaced by an atom or group selected from the group consisting of a
hydrogen atom, a fluorine atom and a methoxy group. [12] The aptamer
according to any of the above-mentioned [1] to [11], which inhibits
neurite outgrowth activity and/or cell proliferation activity of NGF.
[13] A pharmaceutical composition comprising the aptamer according to any
of the above-mentioned [1] to [12]. [14] An anti-pain agent comprising
the aptamer according to any of the above-mentioned [1] to [12]. [15] An
anti-inflammatory agent comprising the aptamer according to any of the
above-mentioned [1] to [12]. [16] A method of treating or preventing a
disease accompanying a pain or inflammation, comprising administering the
aptamer according to any of the above-mentioned [1] to [12] to a subject
in need thereof. [17] The aptamer according to any of the above-mentioned
[1] to [12] for the prophylaxis or treatment of a disease accompanying a
pain or inflammation.

Effect of the Invention

[0026] Since the aptamer or nucleic acid of the present invention shows a
superior NGF inhibitory activity, particularly a high neurite outgrowth
inhibitory activity, due to the above-mentioned constitution, it can be
useful as a medicament for diseases such as algia, inflammatory disease
and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 is a schematic diagram of a predictable secondary structure
of the NGF aptamer shown by SEQ ID NO: 3, wherein the stem-loop structure
on the upper left side corresponds to a consensus secondary structure 1.

[0029] FIG. 3 is a sensorgram showing that the NGF aptamer shown by SEQ ID
NO: 82(2) (modified form) binds to NGF, NT-3 and NT-4, wherein RU on the
vertical axis shows a relative unit, Resp.Diff. shows response
differences, and the horizontal axis shows time (seconds) (Time(s)).
These notations on the vertical axis and the horizontal axis are the same
in the following FIGS. 4-5.

[0032] The present invention provides an aptamer binding to NGF and
capable of forming a potential secondary structure represented by the
formula (I):

##STR00003##

wherein N is one nucleotide selected from the group consisting of A, G,
C, U and T,

[0033] N11-N13, N21-N23, N32-N38 and N42-N48 are the same or different and
each is a bond or 1 or 2 nucleotides selected from the group consisting
of A, G, C, U and T,

[0034] N14, N24, N31, N41, N39 and N49 are the same or different and each
is one nucleotide selected from the group consisting of A, G, C, U and T,

[0035] N14 and N24, N31 and N41, and N39 and N49 each form a Watson-Crick
base pair,

[0036] N11-N12-N13-N14 and N21-N22-N23-N24 are nucleotide sequences
capable of forming a stem structure in combination, and

[0037] N31-N32-N33-N34-N35-N36-N37-N38-N39 and
N41-N42-N43-N44-N45-N46-N47-N48-N49 are nucleotide sequences capable of
forming a stem structure in combination (hereinafter to be described as
"the aptamer of the present invention"). The above-mentioned nucleotide
sequence is optionally modified as mentioned below.

[0038] An aptamer refers to a nucleic acid molecule having a binding
activity for a particular target molecule. The aptamer can inhibit the
activity of a particular target molecule by binding to the particular
target molecule. The aptamer of the present invention is an aptamer
having a binding activity to NGF. According to preferable embodiment, the
aptamer of the present invention can inhibit the activity of NGF by
binding to NGF and inhibiting the binding of NGF and NGF receptor.

[0039] The aptamer of the present invention can be a nucleic acid such as
an RNA, a DNA, a modified nucleic acid or a mixture thereof. Accordingly,
the aptamer of the present invention may be indicated as "the nucleic
acid of the present invention" in the following.

[0040] The single-stranded nucleic acid can have various secondary
structures. The "potential secondary structure" means a secondary
structure that a certain single-stranded nucleic acid can take
thermodynamically in view of its primary structure. Particularly, the
potential secondary structure of the aptamer of the present invention is
a secondary structure predictable using the MFOLD program described in
Example 5. Accordingly, even a nucleic acid not currently having a
secondary structure represented by the above-mentioned formula (I) is
encompassed in the aptamer of the present invention, as long as it has a
primary structure capable of forming said secondary structure.

[0041] Therefore, preferably, the aptamer of the present invention is a
nucleic acid molecule capable of having a secondary structure represented
by the above-mentioned formula (I) thermodynamically stably in view of
the primary structure thereof. In this sense, the aptamer of the present
invention is an aptamer capable of forming a potential secondary
structure represented by the formula (I).

[0042] The potential secondary structure represented by the formula (I) is
what is called a "stem-loop structure", which is particularly a structure
having a loop structure (to be described as "internal loop 1" in the
present Description) between the stem structure that can be formed by a
combination of N11-N12-N13-N14 and N21-N22-N23-N24, and the stem
structure that can be formed by a combination of
N31-N32-N33-N34-N35-N36-N37-N38-N39 and
N41-N42-N43-N44-N45-N46-N47-N48-N49, and further, a loop structure
between N39 and N49 (to be described as "loop 2" in the present
Description).

[0043] The "stem structure" is a structure wherein partial nucleotide
sequences having complementarity in a nucleic acid molecule form
Watson-Crick base pairs (G-C or A-U/T). In the present Description,
N11-N12-N13-N14 and N21-N22-N23-N24, and
N31-N32-N33-N34-N35-N36-N37-N38-N39 and
N41-N42-N43-N44-N45-N46-N47-N48-N49 do not need to be completely
complementary, and mismatch and/or wobbling of G-U/T are/is permitted.
That is, as long as the nucleotides on the both ends of a partial
nucleotide sequence forming a stem structure form Watson-Crick base
pairs, all other nucleotides are not necessarily required to form
Watson-Crick base pairs.

[0044] In the formula (I), "N" positioned in the loop 2 section is one
nucleotide selected from the group consisting of A, G, C, U and T. In a
preferable embodiment, "N" can be G.

[0045] In the formula (I), N11-N13, N21-N23, N32-N38 and N42-N48 are the
same or different and each is a bond or 1 or 2 nucleotides selected from
the group consisting of A, G, C, U and T. When Ni (i is an integer
selected from 11-13, 21-23, 32-38, 42-48) shows "two nucleotides", said
two nucleotides may be the same or different. When Ni shows "two
nucleotides" or "a bond", it is preferably contained in each partial
sequence of N11-N13, N21-N23, N32-N38 and N42-N48 in the number of not
more than 2, more preferably not more than 1. Therefore, each of N11-N14
and N21-N24 forming one stem structure preferably has a nucleotide length
of 2-6, more preferably 3-5, and each of N31-N39 and N41-N49 forming the
other stem structure preferably has a nucleotide length of preferably
7-11, more preferably 8-10.

[0046] The above-mentioned "bond" means a single bond, and when any Ni in
the formula (I) is "a bond", it means that the nucleotides adjacent to
the nucleotide are linked to each other via a phosphodiester bond.

[0047] Particularly preferably, N11-N13, N21-N23, N32-N38 and N42-N48 are
the same or different and each is one nucleotide selected from the group
consisting of A, G, C, U and T. Therefore, the stem structure including
the both ends of the part forming the secondary structure has a stem
length of preferably not more than 4 nucleotides, and the internal stem
structure sandwiched between two loops has a stem length of preferably 9
nucleotides.

[0048] In the formula (I), N14, N24, N31, N41, N39 and N49 are the same or
different and each is one nucleotide selected from the group consisting
of A, G, C, U and T, and N14 and N24, N31 and N41, and N39 and N49 each
form a Watson-Crick base pair (G-C or A-U/T). Therefore, a stem structure
containing the both ends of the part forming the secondary structure
forms a base pair at least at the end on the side of the internal loop 1
section, and the internal stem structure sandwiched between the two loops
forms base pairs at the both ends thereof. More preferably, N14 is U, N24
is A, N31 is G, N41 is C, N39 is G, and N49 is C.

[0049] On the other hand, as defined above, N11-N12-N13 and N21-N22-N23,
and N32-N33-N34-N35-N36-N37-N38 and N42-N43-N44-N45-N46-N47-N48 in the
"stem structure" do not need to be completely complementary (formation of
Watson-Crick base pairs by all of them is not necessary). However,
complementarity of the level enabling formation of a stem structure (loop
(bubble) is not formed in the stem) is necessary. To be specific, since a
loop can be formed when 3 continuous mismatches or wobblings of G-U/T are
contained in each stem structure, each stem structure is desirably free
of 3 continuous mismatches or wobblings of G-U/T. It is also desirable
that not less than 50%, preferably not less than 60%, more preferably not
less than 70%, of each of N11-N12-N13 and N21-N22-N23, and
N32-N33-N34-N35-N36-N37-N38 and N42-N43-N44-N45-N46-N47-N48 be
nucleotides that form Watson-Crick base pairs.

[0050] The present invention also provides a nucleic acid consisting of
the nucleotide sequence of the following (a) or (b):

[0051] Such nucleic acids can form a potential secondary structure
represented by the above-mentioned formula (I).

[0052] While any uracil on any sequence can be replaced by thymine, the
uracil to be replaced can be preferably one in a part other than the
internal loop 1 section and loop 2 section in the aforementioned
potential secondary structure, so that the activity of the aptamer of the
present invention can be maintained.

[0053] In the present Description, a sequence specified by "SEQ ID NO"
means a nucleotide sequence of each aptamer or nucleic acid and, for
example, "a nucleic acid comprising the sequence shown by SEQ ID NO: 1"
means a natural nucleic acid or modified nucleic acid comprising the
sequence shown by SEQ ID NO: 1 or a nucleic acid constituted with the
both. The base sequence of SEQ ID NO of each aptamer is described in the
Sequence Listing.

[0054] In the above-mentioned (b), the number of the nucleotides
substituted, deleted, inserted or added is, for example, about 1-10,
preferably 1-6, more preferably 1-5, further preferably 1-3, most
preferably 1 or 2.

[0055] In the above-mentioned (b), while the position of the nucleotide to
be substituted, deleted, inserted or added is not particularly limited,
the nucleotide can be preferably in a part other than the internal loop 1
section and loop 2 section in the aforementioned potential secondary
structure, so that the activity of the aptamer of the present invention
can be maintained.

[0056] While the nucleotide length of the aptamer or nucleic acid of the
present invention is not particularly limited, it is generally 34--about
200 nucleotides, preferably 34--about 100 nucleotides, more preferably
36-60 nucleotides, further preferably 38-44 nucleotides. The base length
of the aptamer or nucleic acid of the present invention is preferably not
more than 50, more preferably not more than 44. The chemical syntheses
and mass-production of the aptamer become easier by reducing the total
number of nucleotides to fall within the range permitting formation of
the potential secondary structure represented by above-mentioned formula
(I), and there is a major advantage in terms of cost. Such aptamer is
also considered to permit easy chemical modification, high stability in
the body, and low toxicity.

[0057] The aptamer of the present invention can also be a conjugate
selected from the group consisting of a conjugate of a plurality of
nucleic acids consisting of the nucleotide sequence of the
above-mentioned (a), a conjugate of a plurality of nucleic acids
consisting of the nucleotide sequence of the above-mentioned (b), and a
conjugate of a plurality of nucleic acids consisting of the nucleotide
sequence of the above-mentioned (a) and nucleic acids consisting of the
nucleotide sequence of the above-mentioned (b).

[0058] These conjugates can also bind to NGF and/or inhibit the activity
of NGF (NGF receptor binding activity etc.).

[0059] Conjugation herein can be achieved by tandem binding. In the
conjugation, a linker may be utilized. As the linker, nucleotide chains
(e.g., 1 to about 20 nucleotides) and non-nucleotide chains (e.g.,
--(CH2)n-- linker, --(CH2CH2O)n-- linker,
hexaethylene glycol linker, TEG linker, peptide-containing linker,
--S--S-- bond-containing linker, --CONH-- bond-containing linker,
--OPO3-- bond-containing linker) can be mentioned. The plurality as
mentioned in the above-described conjugate of a plurality thereof is not
particularly limited, as long as it is two or more, and the plurality can
be, for example, 2, 3 or 4.

[0060] Each nucleotide contained in the aptamer of the present invention
is the same or different and can be a nucleotide comprising a hydroxyl
group at the 2'-position of ribose (e.g., ribose of pyrimidine
nucleotide, ribose of purine nucleotide) (i.e., unsubstituted nucleotide)
or a nucleotide wherein a hydroxyl group is replaced by any atom or group
at the 2'-position of ribose. As examples of any such atom or group, a
nucleotide substituted by a hydrogen atom, a fluorine atom or an
--O-alkyl group (e.g., --O-Me group), an --O-acyl group (e.g., --O--CHO
group), or an amino group (e.g., --NH2 group) can be mentioned. In
the following cases, the hydroxyl group is replaced by a hydrogen atom, a
fluorine atom or --O-Me group, respectively, at the 2'-position of
ribose.

##STR00004##

[0061] The aptamer of the present invention can also be the nucleotide
wherein at least one kind (e.g., 1, 2, 3 or 4 kinds) of nucleotide
comprises a hydroxyl group, or the above-described any atom or group, for
example, at least two kinds (e.g., 2, 3 or 4 kinds) of groups selected
from the group consisting of a hydrogen atom, a fluorine atom, a hydroxyl
group and a --O-Me group, at the 2'-position of ribose.

[0062] Also, in the aptamer of the present invention, all pyrimidine
nucleotides are the same or different and each can be a nucleotide
substituted by a fluorine atom, or a nucleotide substituted by any atom
or group mentioned above, preferably an atom or group selected from the
group consisting of a hydrogen atom, a hydroxyl group and a methoxy group
at the 2'-position of ribose.

[0063] In the aptamers of the present invention, moreover, all purine
nucleotides are the same or different and each can be a nucleotide
substituted by a hydroxyl group, or a nucleotide substituted by any atom
or group mentioned above, preferably an atom or a group selected from the
group consisting of a hydrogen atom, a methoxy group, and a fluorine atom
at the 2'-position of ribose.

[0064] In the aptamers of the present invention, moreover, all nucleotides
can comprise a hydroxyl group, or any atom or group mentioned above, for
example, the identical group selected by the group consisting of a
hydrogen atom, a fluorine atom, a hydroxyl group and a --O-Me group at
the 2'-position of ribose.

[0065] In this Description, the nucleotides constituting the aptamer are
assumed to be RNAs (i.e., the sugar groups are assumed to be ribose) in
describing how the sugar groups are modified in the nucleotides. However,
this does not mean that DNA is exempted from the aptamer-constituting
nucleotides, and a modification of RNA should read as a modification of
DNA as appropriate. When the nucleotide constituting the aptamer is DNA,
for example, replacement of the hydroxyl group at the 2'-position of
ribose by X should read as a replacement of one hydrogen atom at the
2'-position of deoxyribose by X.

[0066] When uracil is substituted with thymine in the aptamer of the
present invention, NGF-binding activity, NGF-NGF receptor binding
inhibitory activity, NGF neurite outgrowth inhibitory activity, NGF cell
proliferation inhibitory activity, stability, drug deliverability and
stability in blood of the aptamer and the like can be increased.

[0067] The aptamer of the present invention binds to NGF, which is a known
neurotrophin, and is an important secretory protein involved in the
development and survival of peripheral and central neurons. In the
present invention, NGF particularly means a β type NGF. The amino
acid sequences of human β-NGF are those shown by Accession Numbers
NP002497, P01138, AAI26151, AAI26149 and CAB75625, which may also be one
with mutation, its functional domain or peptide fragment. It may be not
only a monomer but also a dimer or multimer. Furthermore, it includes NGF
derived from non-human mammals, for example, primates (e.g., monkey),
rodents (e.g., mouse, rat, guinea pig), and companion animals, domestic
animals and working animals (e.g., dog, cat, horse, bovine, goat, sheep,
swine).

[0068] The aptamer of the present invention inhibits the activity of NGF
by binding to NGF and inhibiting the binding of NGF and NGF receptor. The
aptamer of the present invention may bind to any part of NGF as long as
the binding of NGF and NGF receptor can be inhibited.

[0069] In the present Description, the "inhibitory activity against NGF"
means an inhibitory ability on any activity NGF has. For example, it
means an activity to inhibit NGF from binding to NGF receptor, inhibition
of signal transduction in the downstream of NGF receptor (Ras-MAP kinase
pathway, PI3 kinase pathway), inhibition of increased expression of
TRPV1, SP, BDNF and the like, inhibitory activity of expression of HA,
BK, PG, NGF and other cytokine released from mast cells and the like,
which result from the binding of NGF to NGF receptor, further, inhibition
of differentiation, survival, neurite outgrowth of nerve cell induced by
NGF, increase of blood vessel permeability, enhancement of immune
response of T cells and B cells, differentiation of lymphocytes, growth
and the like of various cells such as mast cells, erythroleukemic cells,
cancer cells and the like, relief of pain, hyperalgesia and the like,
induced by NGF, can be mentioned.

[0070] Preferable "inhibitory activity against NGF" that the aptamer of
the present invention has is an activity to inhibit the binding of NGF to
NGF receptor, an activity to inhibit neurite outgrowth activity induced
by NGF, an activity to inhibit the cell proliferation activity induced by
NGF and the like.

[0071] The aptamer of the present invention binds to NGF in a
physiological buffer (e.g., solution A: see Example 1). The aptamer of
the present invention binds to, for example, NGF at an intensity
detectable by the following test.

[0072] For the measurement, BIAcore2000 manufactured by BIAcore is used.
An aptamer is immobilized on a sensorchip. The amount to be immobilized
is set to 1000 RU. A physiological buffer containing 0.3M NaCl (solution
A: see Example 1) is used to prepare NGF solution (0.5 μM). This NGF
solution (20 μL) is injected and the binding of NGF to the aptamer is
detected. Using RNA containing a random nucleotide consisting of 40
nucleotides as a negative control, when NGF significantly strongly binds
to the aptamer as compared to the control RNA, the aptamer is evaluated
to have bindability to NGF.

[0073] In the present Description, the "NGF receptor" means a cell surface
protein to which NGF binds. As the NGF receptor, TrkA and p75 are known.
The NGF receptor referred to in the present invention may be a protein
containing a natural amino acid sequence or a variant thereof. Here, the
"variant thereof" means a protein or peptide wherein several amino acids
of an amino acid sequence of "NGF receptor" have been substituted, or a
partial amino acid sequence thereof, which has a binding activity to NGF
and inhibits the binding of NGF and an NGF receptor.

[0074] The aptamer of the present invention binds to NGF and inhibits the
binding of NGF and an NGF receptor. Whether or not the aptamer inhibits
the binding of NGF to an NGF receptor, for example, can be evaluated by
the following test.

[0075] For the measurement, BIAcore2000 manufactured by BIAcore is used.
On a CM5 sensorchip is immobilized a fusion protein of NGF receptor and
Fc (e.g., TrkA-Fc (175-TK, R&D systems) or p75-Fc (R&D systems)). The
amount to be immobilized is 500 to 700 RU. NGF (0.1 μM) and an aptamer
(0.2 μM) are mixed in a physiological buffer (solution A: see Example
1), and a mixture to be a sample is prepared over 30 min. This mixture is
injected into BIAcore2000, and the binding of NGF to an NGF receptor is
detected.

[0076] In one embodiment, the aptamer of the present invention can inhibit
both the binding of NGF and TrkA, and that of NGF and p75.

[0077] The aptamer of the present invention is an aptamer that inhibits
neurite outgrowth activity of NGF and/or cell proliferation activity of
NGF. Whether the aptamer of the present invention can inhibit neurite
outgrowth activity of NGF can be evaluated by, for example, the test
described in Example 3. In addition, whether the aptamer of the present
invention can inhibit cell proliferation activity of NGF can be evaluated
by, for example, the test described in Example B.

[0078] The aptamer of the present invention is characterized by the
concentration necessary for inhibiting the neurite outgrowth activity of
NGF by 50% (1050 or 50% inhibitory concentration), which is not more
than 1 nM. Since conventionally-known NGF aptamers have an 1050
value of about several nM for the neurite outgrowth activity of NGF, the
aptamer of the present invention shows more superior property as regards
the neurite outgrowth inhibitory activity.

[0079] In a preferable embodiment, the aptamer of the present invention
also shows an 1050 value of not more than 1 nM for the cell
proliferation activity of NGF.

[0080] On the other hand, whether the aptamer of the present invention has
an activity to inhibit the cell proliferation activity of neurotrophin
other than NGF, specifically, brain-derived neurotrophic factor (BDNF),
neurotrophin-3 (NT-3) and neurotrophin 4/5 (NT-4/5) varies depending on
the aptamer. Here, the terms BDNF, NT-3 and NT-4/5 respectively mean
BDNF, NT-3 and NT-4/5 of all mammals species including human.

[0081] The level of the inhibitory activity against cell proliferation of
other neurotrophins (BDNF, NT-3, NT-4/5) can be evaluated by the test
described in Example 16. The cell proliferation inhibitory activity of
the aptamer of the present invention as described in Example 16 and Table
2 is shown by an 1050 value of not more than 0.1 nM for NGF, and not
less than 1000 nM for BDNF, which means that the aptamer of the present
invention does not inhibit the cell proliferation activity of BDNF.
However, it is 0.97 nM to not less than 10 nM for NT-3; and not more than
3 nM to not less than 30 nM for NT-4. Therefore, inhibition of the cell
proliferation activity of NT-3 and NT-4 varies depending on the aptamers.

[0082] The aptamer of the present invention may be one wherein a sugar
residue (e.g., ribose) of each nucleotide has been modified to increase
the NGF-binding activity, NGF-NGF receptor binding inhibitory activity,
NGF neurite outgrowth inhibitory activity, NGF cell proliferation
inhibitory activity, stability, drug deliverability, and stability in
blood of the aptamer and the like. Examples of the modification in a
sugar residue include replacement of oxygen atom at the 2'-position,
3'-position and/or 4'-position of the sugar residue with another atom,
and the like. As the kind of the modification, fluorination, O-alkylation
(e.g., O-methylation, O-ethylation), O-arylation, S-alkylation (e.g.,
S-methylation, S-ethylation), S-arylation, and amination (e.g.,
--NH2) can be mentioned. In addition, examples thereof include
4'-SRNA wherein the 4'-position oxygen is replaced with sulfur, LNA
(Locked Nucleic Acid) wherein the 2'-position and the 4'-position are
crosslinked via methylene, 3'-N-phosphoramidate nucleic acid wherein the
3'-position hydroxyl group is replaced with an amino group and the like.
Such alterations in the sugar residue can be performed by a method known
per se (see, for example, Sproat et al., (1991) Nucl. Acid. Res. 19,
733-738; Cotton et al., (1991) Nucl. Acid. Res. 19, 2629-2635; Hobbs et
al., (1973) Biochemistry 12, 5138-5145).

[0083] The aptamer of the present invention may also have a nucleic acid
base (e.g., purine or pyrimidine) altered (e.g., chemical substitution)
to increase the NGF-binding activity, NGF-NGF receptor binding inhibitory
activity, NGF neurite outgrowth inhibitory activity, NGF cell
proliferation inhibitory activity, stability, drug deliverability, and
stability in blood of the aptamer and the like. As examples of such
alterations, pyrimidine alteration at 5-position, purine alteration at 6-
and/or 8-position(s) (O-methyl modification and the like), alteration
with an extracyclic amine, substitution with 4-thiouridine, substitution
with 5-bromo or 5-iodo-uracil, modification of 5-amino acid type and
modification of 5-tryptophan side chain can be mentioned. The phosphate
group contained in the aptamer of the present invention may be altered to
confer resistance to nuclease and hydrolysis. For example, the phosphate
region of the aptamer may be replaced with P(O)S (thioate), P(S)S
(dithioate), P(O)NR2 (amidate), P(O)R, P(O)OR', CO or CH2
(formacetal), P(O)BH3 (boranophosphate) or 3'-amine
(--NH--CH2--CH2--) [wherein each unit of R or R' is
independently H or a substituted or unsubstituted alkyl (e.g., methyl,
ethyl)].

[0084] The linking group is, for example, --O--, --N-- or --S--, and
nucleotides can bind to an adjoining nucleotide via these linking groups.

[0085] The alterations may also include alterations such as capping at 3'
and 5'.

[0086] An alteration can further be performed by adding to an end a
polyethyleneglycol (hereinafter, sometimes to be described as "PEG"),
amino acid, peptide, inverted dT, myristoyl, lithocolic-oleyl, docosanyl,
lauroyl, stearoyl, palmitoyl, oleoyl, linoleoyl, other lipids, steroids,
cholesterol, caffeine, vitamins, pigments, fluorescent substances,
anticancer agent, toxin, enzymes, radioactive substance, biotin and the
like. For such alterations, see, for example, U.S. Pat. Nos. 5,660,985
and 5,756,703.

[0087] Particularly, when alteration is performed by terminal addition of
PEG, the molecular weight of PEG is not particularly limited, and is
preferably 1000-100000, more preferably 30000-90000. PEG may be linear or
branched into two or more chains (multi-arm PEG).

[0088] Such PEG is not particularly limited, and those of ordinary skill
in the art can appropriately select and use commercially available or
known PEG (e.g., http://www.peg-drug.com/peg product/branched.html).
Specific preferable examples of the PEG to be applied to the aptamer of
the present invention include 2-branched GS type PEG having a molecular
weight of 40000 (SUNBRIGHT GL2-400GS2 manufactured by NOF CORPORATION),
2-branched TS type PEG having a molecular weight of 40000 (SUNBRIGHT
GL2-400TS manufactured by NOF CORPORATION), 4-branched TS type PEG having
a molecular weight of 40000 (SUNBRIGHT GL4-400TS manufactured by NOF
CORPORATION), 2-branched TS type PEG having a molecular weight of 80000
(SUNBRIGHT GL2-800TS manufactured by NOF CORPORATION), 4-branched TS type
PEG having a molecular weight of 80000 (SUNBRIGHT GL4-800TS manufactured
by NOF CORPORATION) and the like.

[0089] In this case, in the aptamer of the present invention, PEG may be
directly added to the terminus. It is more preferable that a linker
having a group bindable to PEG and the like be added to the terminus
thereof, and PEG be added to the aptamer of the present invention via the
linker.

[0090] The linker for PEG and the aptamer of the present invention is not
particularly limited, and carbon chain number, functional group and the
like can be appropriately selected according to the binding site, the
kind of PEG and the like. Examples of such linker include a linker having
an amino group. Specifically, when added to the 5' end, ssH Linker (SAFC)
or DMS (O)MT-AMINO-MODIFIER (GLEN RESEARCH) can be mentioned, and when
added to the 3' end, TFA Amino C-6 lcaa CPG (ChemGenes) and the like can
be mentioned. When this linker is selected, for example, an active group
of N-hydroxysuccinimide is added to PEG, and reacted with an amino group
on the linker side, whereby the aptamer of the present invention can be
bound to PEG via the linker.

[0091] As PEG and linker, commercially available products can be
preferably used. The reaction conditions and the like relating to the
binding of PEG, a linker and the aptamer of the present invention can be
appropriately determined by those of ordinary skill in the art.

[0092] The aptamer of the present invention can be chemically synthesized
as disclosed herein and by a method known per se in the art. An aptamer
binds to the target substance in a wide variety of binding modes, such as
ionic bonds based on the negative charge of the phosphate group,
hydrophobic bonds and hydrogen bonds based on ribose, and hydrogen bonds
and stacking interaction based on nucleic acid bases. In particular,
ionic bonds based on the negative charge of the phosphate group, which
are present in the same number as the number of constituent nucleotides,
are strong, and bind to the positive charge of lysine and arginine
present on the surface of protein. For this reason, nucleic acid bases
not involved in the direct binding to the target substance can be
substituted. In particular, because the section of stem structure has
already formed base pairs and faces the inside of the double helical
structure, nucleic acid bases are unlikely to bind directly to the target
substance. Therefore, even when a base pair is substituted with another
base pair, the activity of the aptamer often does not decrease. In
structures wherein no base pairs are formed, such as loop structures,
provided that the nucleic acid base is not involved in the direct binding
to the target molecule, base substitution is possible. Regarding
modifications of the 2'-position of ribose, the functional group at the
2'-position of ribose infrequently interacts directly with the target
molecule, but in many cases, it is of no relevance, and can be
substituted by another modified molecule. Hence, an aptamer, unless the
functional group involved in the direct binding to the target molecule is
substituted or deleted, often retains the activity thereof. It is also
important that the overall three-dimensional structure does not change
substantially.

[0093] An aptamer can be prepared by utilizing the SELEX method or an
improved version thereof (e.g., Ellington et al., (1990) Nature, 346,
818-822; Tuerk et al., (1990) Science, 249, 505-510). In the SELEX
method, by increasing the number of rounds or using a competing
substance, an aptamer exhibiting a stronger binding potential for the
target molecule is concentrated and selected. Hence, by adjusting the
number of rounds of SELEX and/or changing the competitive condition,
aptamers with different binding forces, aptamers with different binding
modes, and aptamers with the same binding force or binding mode but
different base sequences can be obtained in some cases. The SELEX method
comprises a process of amplification by PCR; by causing a mutation by
using manganese ions and the like in the process, it is possible to
perform SELEX with higher diversity.

[0094] The aptamers obtained by SELEX are nucleic acids that exhibit high
affinity for the target substance, but this does not mean binding to an
active site of the target substance. Therefore, the aptamers obtained by
SELEX do not necessarily act on the function of the target substance. NGF
is a basic protein, and is thought to be likely to allow nucleic acids to
bind thereto nonspecifically. An aptamer that does not bind to an active
site does not influence the activity of the target substance.

[0095] Based on an active aptamer thus selected, SELEX can be performed
based on the sequence of the obtained aptamer to acquire an aptamer
possessing higher activity. Specifically, after preparing a template
wherein an aptamer with a determined sequence is partially randomized or
a template doped with about 10 to 30% of random sequences, SELEX is
performed again.

[0096] An aptamer obtained by SELEX has a length of about 80 nucleotides,
and this is difficult to prepare as a medicament as it is. Hence, it is
necessary to repeat try-and-error efforts to shorten the aptamer to a
length of about 60 nucleotides or less enabling easy chemical synthesis,
more preferably about 50 nucleotides or less, most preferably 45
nucleotides or less. Depending on the primer design for an aptamer
obtained by SELEX, the ease of the subsequent minimization operation
changes. Unless the primer is designed successfully, subsequent
development will be impossible even if an aptamer with activity is
selected by SELEX.

[0097] Aptamers are altered easily since they permit chemical synthesis.
For aptamers, by predicting the secondary structure using the MFOLD
program, or by predicting the steric structure by X-ray analysis or NMR
analysis, it is possible to predict to some extent which nucleotide can
be substituted or deleted, and where to insert a new nucleotide. A
predicted aptamer with the new sequence can easily be chemically
synthesized, and it can be determined whether or not the aptamer retains
the activity using an existing assay system.

[0098] If a region important to the binding of the obtained aptamer with
the target substance is identified by repeated try-and-error efforts as
described above, the activity remains unchanged in many cases even when a
new sequence is added to both ends of the sequence. Such length of the
new sequence is not particularly limited.

[0099] Modifications, like sequences, afford a wide range of design or
alterations.

[0100] As stated above, aptamers permit a wide range of design or
alterations. The present invention also provides a production method of
aptamer that enables a wide range of design or alteration of an aptamer
comprising a specified sequence (e.g., a sequence corresponding to a
portion selected from among stem section, internal loop section, hairpin
loop section and single-strand section: hereinafter, abbreviated as fixed
sequence as required).

[0101] For example, the production method of such aptamer includes
production of an aptamer comprising a fixed sequence by using a single
kind of nucleic acid molecule consisting of a nucleotide sequence shown
by:

##STR00005##

[wherein (N)a represents a nucleotide chain consisting of "a" units of N;
(N)b represents a nucleotide chain consisting of "b" units of N; each of
the units of N, whether identical or different, is a nucleotide selected
from the group consisting of A, G, C, U and T (preferably, A, G, C and
U). Each of "a" and "b", whether identical or different, can be any
numbers, and can be, for example, 1 to about 100, preferably 1 to about
50, more preferably 1 to about 30, still more preferably 1 to about 20 or
1 to about 10], or plural kinds of nucleic acid molecules (e.g., library
of nucleic acid molecule different in the number of a, b etc.) and primer
pairs corresponding to the primer sequences (i) and (ii), respectively.

[0102] The aptamer of the present invention is preferably an aptamer that
binds to NGF, characteristically contains the sequence shown by SEQ ID
NO: 82, and has a nucleotide length of not more than 50.

[0103] The sequence shown by SEQ ID NO: 82 is a region important for the
aptamer of the present invention to function as the aptamer of the
present invention such as binding to NGF and inhibiting the activity of
NGF, particularly neurite outgrowth activity, cell proliferation activity
and the like. Even when a new sequence is added to both ends of the
sequence, the function of the aptamer of the present invention is not
impaired. The sequence may be subject to modification of the
aforementioned sugar residue, alteration of nucleic acid base and
phosphate group, and the like.

[0104] Thus, preferable specific examples of the aptamer of the present
invention include

aptamers comprising the sequence shown by SEQ ID NO: 82, having a
nucleotide length of not more than 50, and binding to NGF, which are (i)
an aptamer comprising at least one kind of nucleotide wherein the
hydroxyl group is replaced by a hydrogen atom, a fluorine atom, a
--O-alkyl group, a --O-acyl group or an amino group at the 2'-position of
ribose; (ii) an aptamer wherein PEG, amino acid, peptide, inverted dT,
myristoyl, lithocolic-oleyl, docosanyl, lauroyl, stearoyl, palmitoyl,
oleoyl, linoleoyl, other lipid, steroid, cholesterol, caffeine, vitamin,
dye, a fluorescent substance, an anti-cancer agent, a toxin, an enzyme, a
radioactive substance or biotin is added to the terminus; (iii) an
aptamer that satisfies the requirements of (i) and (ii); and the like.

[0105] The present invention also provides a complex comprising the
aptamer of the present invention and a functional substance bound
thereto. The bond between the aptamer and the functional substance in the
complex of the present invention can be a covalent bond or a non-covalent
bond. The complex of the present invention can be one wherein the aptamer
of the present invention and one or more (e.g., 2 or 3) of functional
substances of the same kind or different kinds are bound together. The
functional substance is not particularly limited, as far as it newly
confers a certain function to an aptamer of the present invention, or is
capable of changing (e.g., improving) a certain characteristic which an
aptamer of the present invention can possess. As examples of the
functional substance, proteins, peptides, amino acids, lipids, sugars,
monosaccharides, polynucleotides, and nucleotides can be mentioned. As
examples of the functional substance, affinity substances (e.g., biotin,
streptavidin, polynucleotides possessing affinity for target
complementary sequence, antibodies, glutathione Sepharose, histidine),
substances for labeling (e.g., fluorescent substances, luminescent
substances, radioisotopes), enzymes (e.g., horseradish peroxidase,
alkaline phosphatase), drug delivery vehicles (e.g., liposome,
microspheres, peptides, polyethyleneglycols), drugs (e.g., those used in
missile therapy such as calicheamycin and duocarmycin; nitrogen mustard
analogues such as cyclophosphamide, melphalan, ifosfamide or
trofosfamide; ethylenimines such as thiotepa; nitrosoureas such as
carmustine; alkylating agents such as temozolomide or dacarbazine;
folate-like metabolic antagonists such as methotrexate or raltitrexed;
purine analogues such as thioguanine, cladribine or fludarabine;
pyrimidine analogues such as fluorouracil, tegafur or gemcitabine; vinca
alkaloids such as vinblastine, vincristine or vinorelbine and analogues
thereof; podophyllotoxin derivatives such as etoposide, taxans, docetaxel
or paclitaxel; anthracyclines such as doxorubicin, epirubicin, idarubicin
and mitoxantrone, and analogues thereof; other cytotoxic antibiotics such
as bleomycin and mitomycin; platinum compounds such as cisplatin,
carboplatin and oxaliplatin; pentostatin, miltefosine, estramustine,
topotecan, irinotecan and bicalutamide), and toxins (e.g., ricin toxin,
liatoxin and Vero toxin) can be mentioned. These functional molecules are
finally removed in some cases. Furthermore, the molecules may be peptides
that can be recognized and cleaved by enzymes such as thrombin, matrix
metalloproteinase (MMP), and Factor X, and may be polynucleotides that
can be cleaved by nucleases or restriction endonuclease.

[0106] The aptamer or the complex of the present invention can be used as,
for example, a medicament or a diagnostic agent, a test drug, a reagent,
an additive for drinking water and food, an enhancer and a mitigator.

[0107] The aptamer and complex of the present invention can have an
activity to inhibit the function of NGF by binding to NGF and inhibiting
the binding of NGF and an NGF receptor. As mentioned above, NGF is deeply
involved in the pain and inflammation. Therefore, the aptamer and complex
of the present invention are useful as medicaments for the prophylaxis or
treatment of diseases accompanying pain or inflammation (anti-pain agent,
anti-inflammatory agent etc.).

[0109] While the disease associated with inflammation here is not
particularly limited, systemic lupus erythematosus, multiple sclerosis,
psoriasis, osteoarthritis, rheumatoid arthritis, interstitial cystitis,
asthma and the like can be mentioned.

[0111] When NGF binds to a receptor thereof, TrkA, it activates tyrosine
phosphorylation of TrkA and Ras-MAPK, PLC-γ, PI3K and the like at
the downstream of TrkA, and exhibits physiological actions such as
survival and differentiation of nerve cells. On the other hand, it
induces cell death in the signal pathway via p75 receptor. Therefore, the
aptamer and complex of the present invention can be used as medicaments,
diagnostic agents, test drugs, or reagents for diseases relating to
activation of these signal transduction pathways. Examples of the
diseases relating to the activation of these signal transduction pathways
include the above-mentioned algia, inflammatory disease and cancers.

[0112] When the aptamer and complex of the present invention are used as
medicaments, diagnostic agents, test drugs, reagents and the like, the
subject of administration thereof is not particularly limited and, for
example, primates (e.g., human, monkey), rodents (e.g., mouse, rat,
guinea pig), and companion animals, domestic animals and working animals
(e.g., dog, cat, horse, bovine, goat, sheep, swine) can be mentioned.

[0113] The aptamer and complex of the present invention are capable of
binding specifically to NGF. Therefore, the aptamer and complex of the
present invention are useful as probes for NGF detection. The probes are
useful in in vivo imaging of NGF, measurements of blood concentrations,
tissue staining, ELISA and the like. The probes are also useful as
diagnostic agents, test drugs, reagents and the like for diseases
involving NGF (diseases accompanied by pain or inflammation, and the
like).

[0114] Based on their specific binding to NGF, the aptamer and complex of
the present invention can be used as ligands for separation and
purification of NGF.

[0115] In addition, the aptamer and complex of the present invention can
be used as test drugs for examining the mental condition of romance and
the like, or medicaments, regulators, enhancers or mitigators for
controlling the mental condition.

[0116] The aptamer and complex of the present invention can be used as
drug delivery vehicles.

[0117] The medicament of the present invention can be one formulated with
a pharmaceutically acceptable carrier. As examples of the
pharmaceutically acceptable carrier, excipients such as sucrose, starch,
mannit, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, and
calcium carbonate; binders such as cellulose, methylcellulose,
hydroxylpropylcellulose, polypropylpyrrolidone, gelatin, gum arabic,
polyethylene glycol, sucrose, and starch; disintegrants such as starch,
carboxymethylcellulose, hydroxylpropylstarch, sodium-glycol-starch,
sodium hydrogen carbonate, calcium phosphate, and calcium citrate;
lubricants such as magnesium stearate, Aerosil, talc, and sodium lauryl
sulfate; flavoring agents such as citric acid, menthol,
glycyrrhizin-ammonium salt, glycine, and orange powder; preservatives
such as sodium benzoate, sodium hydrogen sulfite, methylparaben, and
propylparaben; stabilizers such as citric acid, sodium citrate, and
acetic acid; suspending agents such as methylcellulose,
polyvinylpyrrolidone, and aluminum stearate; dispersing agents such as
surfactants; diluents such as water, physiological saline, and orange
juice; base waxes such as cacao butter, polyethylene glycol, and
kerosene; and the like can be mentioned, but these are not limitative.

[0118] Preparations suitable for oral administration are a solution
prepared by dissolving an effective amount of ligand in a diluent such as
water, physiological saline, or orange juice; capsules, sachets or
tablets comprising an effective amount of ligand in solid or granular
form; a suspension prepared by suspending an effective amount of active
ingredient in an appropriate dispersant; an emulsion prepared by
dispersing and emulsifying a solution of an effective amount of active
ingredient in an appropriate dispersant, and the like.

[0119] The medicament of the present invention can be coated by a method
known per se for the purpose of taste masking, enteric dissolution,
sustained release and the like as necessary. As examples of coating
agents used for the coating, hydroxypropylmethylcellulose,
ethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose,
polyoxyethylene glycol, Tween 80, Pluronic F68, cellulose acetate
phthalate, hydroxypropylmethylcellulose phthalate, hydroxymethylcellulose
acetate succinate, Eudragit (manufactured by Rohm, Germany, methacrylic
acid/acrylic acid copolymer), pigments (e.g., ferric oxide red, titanium
dioxide and the like) and the like are used. The medicament may be a
rapid-release preparation or sustained-release preparation. Examples of
sustained-release bases include liposome, atelocollagen, gelatin,
hydroxyapatite, PLGA and the like.

[0120] As preparations suitable for parenteral administration (e.g.,
intravenous administration, subcutaneous administration, intramuscular
administration, topical administration, intraperitoneal administration,
intranasal administration, pulmonary administration and the like),
aqueous and non-aqueous isotonic sterile injectable liquids are
available, which may comprise an antioxidant, a buffer solution, a
bacteriostatic agent, an isotonizing agent and the like. Aqueous and
non-aqueous sterile suspensions can also be mentioned, which may comprise
a suspending agent, a solubilizer, a thickener, a stabilizer, an
antiseptic and the like. The preparation can be included in a container
such as an ampoule or a vial in a unit dosage volume or in several
divided doses. An active ingredient and a pharmaceutically acceptable
carrier can also be freeze-dried and stored in a state that may be
dissolved or suspended in an appropriate sterile vehicle just before use.
Sustained-release preparations are also suitable preparations. The
sustained-release preparations include sustained release from carriers or
containers embedded in the body, such as artificial bones, biodegradable
or non-degradable sponges, bags, drug pumps, osmotic pressure pumps and
the like. Devices for continuous or intermittent, systemic or topical
delivery from outside the body are also included in the scope of
sustained-release preparations. Biodegradable bases include liposome,
cationic liposome, poly(lactic-co-glycolic) acid (PLGA), atherocollagen,
gelatin, hydroxyapatite, polysaccharide sizofiran. In addition to liquid
injections and sustained release preparation, inhalants and ointments are
also acceptable. In the case of an inhalant, an active ingredient in a
freeze-dried state is micronized and administered by inhalation using an
appropriate inhalation device. An inhalant can be formulated as
appropriate with a conventionally used surfactant, oil, seasoning,
cyclodextrin or derivative thereof and the like as required.

[0122] An inhalant can be produced according to a conventional method.
Specifically, an inhalant can be produced by powdering or liquefying the
above-described aptamer and complex of the present invention, blending it
in an inhalation propellant and/or carrier, and filling them in an
appropriate inhalation vessel. When the above-described aptamer and
complex of the present invention is a powder, an ordinary mechanical
powder inhalator can be used; in the case of a liquid, an inhalator such
as a nebulizer can be used. Here, as the propellant, conventionally known
one can be widely used; chlorofluorocarbon-series compounds such as
chlorofluorocarbon-11, chlorofluorocarbon-12, chlorofluorocarbon-21,
chlorofluorocarbon-22, chlorofluorocarbon-113, chlorofluorocarbon-114,
chlorofluorocarbon-123, chlorofluorocarbon-142c, chlorofluorocarbon-134a,
chlorofluorocarbon-227, chlorofluorocarbon-C318, and
1,1,1,2-tetrafluoroethane, hydrocarbons such as propane, isobutane, and
n-butane, ethers such as diethyl ether, compressed gases such as nitrogen
gas and carbon dioxide gas and the like can be mentioned.

[0123] The dosage of the medicament of the present invention varies
depending on the kind and activity of active ingredient, seriousness of
disease, animal species being the subject of administration, drug
tolerability of the subject of administration, body weight, age and the
like, and the usual dosage, based on the amount of active ingredient per
day for an adult, can be about 0.0001 to about 100 mg/kg, for example,
about 0.0001 to about 10 mg/kg, preferably about 0.005 to about 1 mg/kg.

[0124] The present invention also provides a solid phase carrier having
the aptamer and the complex of the present invention immobilized thereon.
As examples of the solid phase carrier, a substrate, a resin, a plate
(e.g., multiwell plate), a filter, a cartridge, a column, and a porous
material can be mentioned. The substrate can be one used in DNA chips,
protein chips and the like; for example, nickel-PTFE
(polytetrafluoroethylene) substrates, glass substrates, apatite
substrates, silicone substrates, alumina substrates and the like, and
substrates prepared by coating these substrates with a polymer and the
like can be mentioned. As examples of the resin, agarose particles,
silica particles, a copolymer of acrylamide and
N,N'-methylenebisacrylamide, polystyrene-crosslinked divinylbenzene
particles, particles of dextran crosslinked with epichlorohydrin,
cellulose fiber, crosslinked polymers of aryldextran and
N,N'-methylenebisacrylamide, monodispersed synthetic polymers,
monodispersed hydrophilic polymers, Sepharose, Toyopearl and the like can
be mentioned, and also resins prepared by binding various functional
groups to these resins were included. The solid phase carrier of the
present invention can be useful in, for example, purifying, detecting and
quantifying NGF.

[0125] The aptamer and the complex of the present invention can be
immobilized onto a solid phase carrier by a method known per se. For
example, a method that introduces an affinity substance (e.g., those
described above) or a predetermined functional group into the aptamer or
the complex of the present invention, and then immobilizes the aptamer
and complex onto a solid phase carrier via the affinity substance or
predetermined functional group can be mentioned. The present invention
also provides such methods. The predetermined functional group can be a
functional group that can be subjected to a coupling reaction; for
example, an amino group, a thiol group, a hydroxyl group, and a carboxyl
group can be mentioned. The present invention also provides an aptamer
having such a functional group introduced thereto.

[0126] The present invention also provides a method of purifying and
concentrating NGF. In particular, the present invention makes it possible
to separate NGF from the proteins of other family proteins. The method of
purification and concentration of the present invention can comprise
adsorbing NGF to the solid phase carrier of the present invention, and
eluting the adsorbed NGF with an eluent. Adsorption of NGF to the solid
phase carrier of the present invention can be achieved by a method known
per se. For example, a NGF-containing sample (e.g., bacterial or cell
culture or culture supernatant, blood) is introduced into the solid phase
carrier of the present invention or a composition containing the same.
NGF can be eluted using an eluent such as a neutral solution. There is no
limitation on the neutral eluent, which can have a pH of, for example,
about 6 to about 9, preferably about 6.5 to about 8.5, and more
preferably about 7 to about 8. The neutral solution can also comprise,
for example, urea, a chelating agent (e.g., EDTA), a potassium salt
(e.g., KCl), a magnesium salt (e.g., MgCl2), a surfactant (e.g.,
Tween 20, Triton, NP40), and glycerin. The method of purification and
concentration of the present invention can further comprise washing the
solid phase carrier using a washing solution after NGF adsorption.
Examples of the washing solution include those containing urea, a
chelating agent (e.g., EDTA), Tris, an acid, an alkali, Transfer RNA,
DNA, surfactants such as Tween 20, salts such as NaCl and the like. The
method of purification and concentration of the present invention can
still further comprise heating the solid phase carrier. This step enables
the regeneration and sterilization of the solid phase carrier.

[0127] The present invention also provides a method of detecting and
quantifying NGF. In particular, the present invention makes it possible
to detect and quantify NGF separately from the proteins of other family
proteins. The method of detection and quantitation of the present
invention can comprise measuring NGF by utilizing the aptamer of the
present invention (e.g., by the use of the complex and solid phase
carrier of the present invention). The method of detecting and
quantifying NGF can be performed in the same manner as an immunological
method, except that the aptamer of the present invention is used in place
of an antibody. Therefore, by using the aptamer of the present invention
as a probe in place of an antibody, in the same manner as such methods as
enzymeimmunoassay (EIA) (e.g., direct competitive ELISA, indirect
competitive ELISA, sandwich ELISA), radioimmunoassay (RIA), fluorescent
immunoassay (FIA), Western blot technique, immunohistochemical staining
method, and cell sorting method, detection and quantitation can be
performed. The aptamer of the present invention can also be used as a
molecular probe for PET and the like. These methods can be useful in, for
example, measuring NGF contents in living organisms or biological
samples, and in diagnosing a disease associated with NGF.

[0128] The disclosures in all publications mentioned herein, including
patents and patent application specifications, are incorporated by
reference herein in the present invention to the extent that all of them
have been given expressly.

[0129] The present invention is hereinafter described in more detail by
means of the following Examples, which, however, never limit the scope of
the invention.

EXAMPLES

Example 1

Production of NGF Aptamer--1

[0130] RNA aptamers that bind specifically to NGF were prepared using the
SELEX method. The SELEX was performed by referring the method of
Ellington et al. (Ellington and Szostak, Nature 346, 818-822, 1990) and
the method of Tuerk et al. (Tuerk and Gold, Science 249, 505-510, 1990).
Human NGF (manufactured by R&D Systems) was used as a target substance.

[0131] The RNA used in the first round (40N) was obtained by transcribing
a chemically synthesized DNA using the DuraScribe® T7 Transcription
Kit (manufactured by Epicentre). Of the NTPs contained in the kit, 2'-OH
ATP was replaced by 2'-deoxyadenosine 5'-triphosphate (2'-H ATP or dATP,
manufactured by GE Healthcare) and other substrates contained in the kit
were used. The RNA obtained by this method has a fluorinated 2'-position
of the ribose of the pyrimidine nucleotide, and G (purine nucleotide) is
of RNA type, and A is of DNA type. The DNA of 83 nucleotides shown below,
having a primer sequence at each end of a 40-nucleotide random sequence
was used as DNA template. The DNA template and the primers were prepared
by chemical synthesis.

[0132] In the above-mentioned sequence, n represents any one of a, g, c
and t. The primer Fwd1 comprises a promoter sequence of T7 RNA
polymerase. The variation of the RNA pool used in the first round was
theoretically 1014.

[0133] After 10 rounds of SELEX, the PCR product was cloned into a pGEM-T
Easy vector (manufactured by Promega), which was used to transform
Escherichia coli strain DH5α (manufactured by Toyobo). The plasmid
was extracted from a single colony and the base sequences were determined
by DNA sequencer (ABI PRISM3100, manufactured by ABI). 48 clones were
examined, and 45 sequences could be sequenced. Among them were 7 kinds of
the same 2 sequences, and the remaining 31 sequences were single
sequences. 3 rounds of SELEX were further added, and the sequence was
examined. As a result, further convergence was observed.

[0134] The sequences showing convergence in 10 and 13 rounds and several
single sequences were evaluated for the binding activity to NGF by a
surface plasmon resonance method.

[0135] As the measuring apparatus, BIAcore2000 manufactured by BIAcore was
used and, as the sensor chip, CM5 that reacts with an amino group was
used. Human NGF was dissolved in immobilization solution (10 mM sodium
acetate, pH 6) at 25-40 μg/ml. For the reaction of an amino group on
the protein side and a carboxyl group on the chip side,
ethyl-3-carbodiimide hydrochloride and N-hydroxysuccinimide were used.
After the reaction, blocking by ethanolamine-HCl was performed. The
immobilized amount of NGF was set to 3,000-4,000 RU. An aptamer for
analyte was prepared to 0.15 μM-0.5 μM. As a running buffer,
solution A was used. Here, solution A is a mixed solution of 145 mM
sodium chloride, 5.4 mM potassium chloride, 1.8 mM calcium chloride, 0.8
mM magnesium chloride, 20 mM Tris (pH 7.6), 0.05% Tween 20. As a
regeneration solution, a mixed solution of 1M NaCl and 50 mM NaOH was
used. NGF was immobilized on FC2, and the results of FC1 were subtracted
to give a final sensorgram.

[0136] The binding activity of 34 sequences was measured to find that all
RNAs more significantly bind to NGF than 40N of the control. Here, 40N
refers to the RNA pool used for the first round, comprising a
40-nucleotide random sequence. From the above, it was shown that these
RNAs are aptamers that bind to NGF.

Example 2

Aptamer Inhibiting Binding of NGF and NGF Receptor

[0137] Whether the aptamers obtained in Example 1 inhibit the binding of
NGF and an NGF receptor (TrkA and p75) was determined using the surface
plasmon resonance method.

[0138] As directed in BIAcore's protocol, Protein A (21181, PIERCE) was
immobilized on a CM5 sensor chip. About 700 to 1200 RU of human TrkA
fused with the Fc portion of IgG (175-TK, R&D systems) or human P75
(367-NR, R&D systems) was immobilized thereon. As the analyte, a mixture
of NGF (0.1 μM) and each aptamer (0.3 μM) was injected after being
allowed to stand for 30 minutes. If the aptamer inhibits the binding of
NGF and TrkA or p75, the signal on the sensorgram is expected to not
rise; if the aptamer does not inhibit the binding, a triple complex will
be formed and the signal is expected to rise. When NGF binds stronger to
a receptor than an aptamer, the aptamer may be removed and NGF may bind
to the receptor. Before starting the inhibition experiment, binding of
TrkA or p75 and NGF was confirmed.

[0139] The inhibitory activity of 34 sequences was measured to find that
all aptamers inhibit the binding of NGF and TrkA or p75. Particularly,
the aptamers shown by SEQ ID NOs: 1, 2, 3, 4, 5, 7 showed a strong
inhibitory activity. From the above, it was shown that these RNAs are
aptamers that inhibit the binding of NGF and NGF receptor.

Example 3

Neurite Outgrowth Inhibitory Activity of Aptamer

[0140] The neurite outgrowth inhibitory activity of the aptamer obtained
in Example 1 was evaluated by using Neuroscreen-1 cell, which is a
subclone of PC-12 cells.

[0141] The cells (2500 cells per well) were cultured for one day in an
RPMI-1640 medium containing 2.5% horse serum and 1.25% fetal bovine serum
in a 96 well flat-bottom plate coated with collagen type IV. A mixed
solution of human NGF (final concentration 0.38 nM or 1.14 nM) and an
aptamer (final concentration 500-0.01 nM), which had been prereacted in a
serum-free RPMI-1640 medium at room temperature or 37° C. for 30
min to 1 hr, was added. Two days later, the cytoplasm and nuclei were
stained using Cellomics Neurite Outgrowth Kit (manufactured by Thermo
Scientific), and neurite length per cell was measured by Cellomics
ArrayScan VTI (manufactured by Thermo Scientific). With the neurite
length per cell obtained by the addition of NGF alone as inhibitory
activity 0%, and that of the cell obtained by NGF free culture for 2 days
as inhibitory activity 100%, the inhibitory activity of the aptamer was
calculated from the neurite length per cell obtained by culturing with
the addition of NGF and the aptamer in mixture.

[0142] The inhibitory activity of the 34 kinds of aptamers obtained in
Example 1 was measured to find that the aptamers shown by SEQ ID NOs: 1-8
strongly inhibit neurite outgrowth when 10 nM thereof is added. Other
aptamers did not show remarkable inhibition at 10 nM.

[0143] The nucleotide sequences actually obtained which correspond to each
SEQ ID NO are shown below. The upper-case letters show RNA, lower-case
letters show DNA, the parentheses in the nucleotides show the
modification at the 2'-position and F is a fluorine atom (hereinafter the
same).

[0144] The aptamers shown by SEQ ID NOs: 3 and 6 were subjected to
shortening. Using MFOLD program (Zuker, Nucleic Acids Res. 31, 3406-3415,
2003), the secondary structure of RNA was predicted and the chain was
shortened while referring to the structure thereof. The shortened form
was obtained by chemically synthesizing the DNA of the object sequence
and transcribing same using DuraScribe T7 Transcription Kit (manufactured
by Epicentre). The transcription product was treated with DNase, the
protein was removed by a phenol-chloroform treatment, and RNA was
collected by ethanol precipitation. The purity of the recovered RNA was
confirmed by polyacrylamide electrophoresis, and the quantity was
confirmed by an absorbance measurement method. The actually produced
sequences in a shortened form are as described below.

[0145] The binding activity of these aptamers to NGF was evaluated by the
surface plasmon resonance method in the same manner as in Example 1. As a
result, RNAs shown by SEQ ID NOs: 9-21 were found to bind more
significantly to NGF than 40N of is the control. On the other hand, the
binding amount of RNAs shown by SEQ ID NOs: 121-141 markedly decreased as
compared to the aptamer shown by SEQ ID NO: 11 or 13.

[0146] The inhibition of the binding of NGF to receptors thereof (TrkA and
p75) was evaluated by the surface plasmon resonance method in the same
manner as in Example 2 to find that the aptamers shown by SEQ ID NOs:
9-16 have a high inhibitory activity.

[0147] The neurite outgrowth inhibitory activity was examined by a method
similar to Example 3 to find that the aptamers shown by SEQ ID NOs: 9-21
show a high inhibitory activity at a concentration of 10 nM. On the other
hand, the aptamers shown by SEQ ID NOs: 127, 128, 131, 133, 135, 141 did
not show a remarkable inhibitory activity at 10 nM.

Example 5

Prediction of Secondary Structure of Aptamer Shown By SEQ ID NO: 3 and
Shortened Form Thereof

[0150] The aptamers shown by SEQ ID NOs: 12 and 13 are altered forms of
the aptamer shown by SEQ ID NO: 11, wherein stem 1 is substituted by G-C
pair. The aptamers shown by SEQ ID NOs: 12 and 13 showed a neurite
outgrowth inhibitory activity equivalent to that of SEQ ID NO: 11.
Therefore, it was suggested that the activity is not markedly influenced
by the base pair as long as stem 1 is a stem structure. On the other
hand, the activity of RNA shown by SEQ ID NO: 129, which is an aptamer
shown by SEQ ID NO: 13 wherein U-a base pair is removed from stem 1,
markedly decreased. Therefore, it was found that the 4th base pair of
stem 1 needs to be U-a.

[0153] The structure specified in FIG. 2 is hereinafter to be referred to
as the consensus secondary structure 1.

Example 6

Production of NGF Aptamer--2

[0154] Using a primer different from that in Example 1, SELEX was
performed, and whether an aptamer having a consensus secondary structure
1 can be obtained was studied. The DNA template and the primer sequences
used are shown below.

[0155] In the above-mentioned sequences, n represents any one of a, g, c
and t. The primer Fwd2 comprises a promoter sequence of T7 RNA
polymerase. The variation of the RNA pool used in the first round was
theoretically 1014.

[0156] SELEX was performed in the same manner as in Example 1. After 10
rounds of SELEX, 48 clones were examined and 46 sequences could be
sequenced. Among them were 1 sequence wherein 5 clones are the same, 4
sequences wherein 3 clones are the same, and 3 sequences wherein 2 clones
are the same, and a total of 8 sequences showed convergence. The
remaining 23 sequences were single sequences.

[0157] 8 sequences showing convergence were selected, and the binding
activity to NGF was evaluated by the surface plasmon resonance method.
The measurement method was similar to Example 1. As a result, all
sequences bound to NGF only slightly.

[0158] The secondary structure of all sequences including single sequences
was predicted using an MFOLD program to find no sequence containing
consensus secondary structure 1.

Example 7

Production of NGF Aptamer--3

[0159] SELEX was performed using an RNA pool containing a sequence shown
by SEQ ID NO: 12 doped with 15% random sequence and added with primer
sequences, similar to those in Example 1, to the both ends thereof. SELEX
was performed almost in the same manner as in Example 1. The sequences of
the template are shown below.

[0161] After the completion of 4 rounds, the sequence of 48 clones was
confirmed to find that about half was a sequence containing SEQ ID NO: 12
and the rest was a sequence with mutations at several sites. To remove
the same sequence as SEQ ID NO: 12, antisense oligo of SEQ ID NO: 12 was
added to the RNA pool, and SELEX was performed for 3 more rounds. The
sequence of antisense oligo is as described below.
5'-agacggaaactacgcagcagga-3'-(SEQ ID NO: 146)

[0162] The antisense oligo was added in a 10-fold amount relative to the
RNA pool. The sequence of the obtained RNA was confirmed to find that
about half was a sequence shown by SEQ ID NO: 12 with mutations at
several sites and the rest was a sequence completely different from the
sequence shown by SEQ ID NO: 12.

[0163] A total of 16 sequences shown by SEQ ID NOs: 22-37 were selected
from 4 and 7 rounds, and the binding activity to NGF and the inhibitory
activity against the binding of NGF and NGF receptor were examined. The
measurement was as shown in Examples 1 and 2 and the surface plasmon
resonance method was used. As a result of the measurement, it was found
that all sequences more significantly bind to NGF than 40N of the
control, and inhibit the binding of NGF and NGF receptor. In addition,
the neurite outgrowth inhibitory activity was measured by a method
similar to that in Example 3. As a result, all sequences showed a high
inhibitory activity at a concentration of 10 nM. The nucleotide sequences
actually obtained, which correspond to each SEQ ID NO, are shown below.

[0164] The secondary structure of the aptamers shown by SEQ ID NOs: 22-37
was predicted using the MFOLD program to find that all aptamers other
than the aptamers shown by SEQ ID NOs: 30, 33, 36 contain consensus
secondary structure 1. The 5' side of all the sequences of the internal
loops 1 was CCU and the 3' side was UGUU (FIG. 2). In addition, loop 2
contained a consensus sequence shown by 5'-UUUCCXU-3'. Here, X is any of
A, G, C and U. All the final base pair of stem 1 was U-a. The 1st, 5th,
6th, 8th and 9th base pairs of stem 2 were G-C, C-G, G-C, a-U and G-C,
respectively. The 2nd-4-th and 7th contained some different base pairs.

Example 8

Cell Proliferation Inhibitory Activity of Aptamer (TF-1 Assay)

[0165] The inhibitory activities of the aptamers shown by SEQ NOs. 22 to
37 were evaluated by a proliferation inhibition assay using TF-1 cells.

[0166] Two NGF receptor (human TrkA and human p75) genes were introduced
into TF-1 cells (ATCC Number: CRL-2003), which is a human erythroleukemic
cell line, by using a retrovirus vector to give cells that highly express
two receptors simultaneously and stably. The cells were suspended in an
RPMI-1640 medium containing 20% fetal bovine serum, and seeded in a white
96 well flat-bottom plate at 1000 cells (50 μL) per well. Thereto was
added a mixed solution 50 μL of human NGF (final concentration 0.076
nM) and the aptamer (final concentration 30-0.01 nM), which had been
pre-reacted at room temperature for 30 min in a serum-free RPMI-1640
medium, 3 days later, 100 μL of CellTiter-Glo reagent for
CeliTiter-Glo Luminescent Cell Viability Assay (manufactured by Promega)
was added to each well, chemiluminescence was measured by a microplate
reader and the growth of TF-1 cells by NGF stimulation was evaluated.
With the amount of luminescence per well obtained by the addition of NGF
alone and culture of the cells for 3 days as inhibitory activity 0%, and
that of the well obtained by NGF free culture for 3 days as inhibitory
activity 100%, the inhibitory activity of the aptamer was calculated from
the amount of luminescence per well obtained by culturing with the
addition of NGF and the aptamer in mixture. As a result, it was found
that all these aptamers show a high inhibitory activity at a
concentration of 10 nM.

[0167] The inhibitory activity of the aptamers shown by SEQ ID NO: 62 and
68 described in WO 2010/035725A1 was examined for comparison. As a
result, IC50 was 6.1 and 7.5 nM, respectively.

Example 9

Shortening of Aptamer--2

[0168] The aptamers shown by SEQ ID NOs: 23, 25, 26, 27, 28, 29, 31, 32,
34 and 35 that showed a high inhibitory activity were subjected to
shortening by reference to the consensus secondary structure 1. The
binding activity to NGF was measured by the surface plasmon resonance
method in the same manner as in Example 1 to find that all shortened
forms strongly bound to NGF. In addition, the neurite outgrowth
inhibitory activity and TF-1 cell proliferation inhibitory activity were
measured by a method similar to that in Examples 3 and B to find a strong
inhibitory activity at a concentration of 10 nM. The nucleotide sequences
actually obtained are shown below.

[0169] The secondary structure of these aptamers was predicted using the
MFOLD program to find that all of them had a structure shown by the
consensus secondary structure 1.

Example 10

Production of NGF Aptamer--4

[0170] SELEX was performed using an RNA pool containing a sequence shown
by SEQ ID NO: 12 doped with 21% random sequence and added with primer
sequences, similar to those in Example 1, to the both ends thereof. SELEX
was performed in the same manner as in Example 1. The sequence of the
template is shown below.

[0172] After the completion of 4 rounds, the sequences of 48 clones were
confirmed, but sequence convergence was not seen. Thus, 3 more rounds
were performed. At 5, 6, 7 rounds, the sequences of 48 clones were
confirmed to find sequence convergence as the rounds proceeded. At 7th
round, almost all sequences showed convergence.

[0173] A total of 14 sequences were selected from the 5, 6, 7 rounds, the
binding activity to NGF was measured by the surface plasmon resonance
method. The measurement method is as shown in Example 1. As a result of
the measurement, all sequences significantly bound to NGF than 40N of the
control. The nucleotide sequences actually obtained are shown below.

[0174] The secondary structure of these aptamers was predicted using the
MFOLD program to find that all of them had a structure shown by the
consensus secondary structure 1. The 5' side of all the sequences of the
internal loops 1 was CCU and the 3' side was UGUU. In addition, loop 2
contained a consensus sequence shown by 5'-UUUCCXU-3'. Here, X is either
G or U. The 1st, 3rd, 8th and 9th base pairs of stem 2 were G-C, U-G, a-U
and G-C, respectively. The 2nd and 4th-7th contained some different base
pairs.

[0175] In the same manner as in Example 4, these aptamers were subjected
to shortening. As a result, in all aptamers, the chain could be shortened
to a structure similar to the consensus secondary structure 1 while
maintaining the binding activity.

[0176] In the same manner as in Examples 3 and 8, the neurite outgrowth
inhibitory activity and TF-1 cell proliferation inhibitory activity were
evaluated. As a result, these aptamers all showed a high inhibitory
activity at a concentration of 10 nM. The nucleotide sequences actually
obtained are shown below.

[0178] After the completion of 7 rounds, the sequences of 48 clones were
confirmed, but sequence convergence was not seen. Thus, 3 more rounds
were performed. After the completion of 10 m rounds, the sequences of 48
clones were confirmed to find convergence in about half the sequences.
The remaining half sequences were single sequences. 17 sequences were
selected therefrom, and the binding activity to NGF was measured by the
surface plasmon resonance method. The measurement method is as shown in
Example 1. As a result of the measurement, it was found that all
sequences more significantly bind to NGF than 40N of the control.

[0179] In the same manner as in Example 4, the above-mentioned aptamers
were subjected to shortening. As a result, in all aptamers, the chain
could be shortened to a structure similar to the consensus secondary
structure 1 while maintaining the binding activity. The nucleotide
sequences actually obtained are shown below.

[0180] With the neurite length per cell obtained by the addition of NGF
alone as inhibitory activity 0%, and that of the cell obtained by NGF
free culture for 2 days as inhibitory activity 100%, the neurite
outgrowth inhibitory activities of these aptamers were calculated from
the neurite length per cell obtained by culturing with the addition of
NGF and the aptamer in mixture. The 50% inhibitory concentration
(IC50) was determined from the concentrations at two, above and
below points sandwiching the 50% inhibitory activity. The experiment
results are shown in Table 1. In Table 1, the IC50 value indicated
as "<X" means that the inhibitory activity was not less than 50% when
the indicated concentration X was minimum measured concentration. All
tested aptamers showed a strong inhibitory activity. The IC50 values
thereof are partly shown in Table 1.

[0181] As regards the TF-1 cell proliferation inhibitory activity, using
the amount of luminescence per well obtained by the addition of NGF alone
and culture of the cells for 3 days as inhibitory activity 0%, and that
of the well obtained by NGF free culture for 3 days as inhibitory
activity 100%, the inhibitory activity of the aptamer was calculated from
the amount of luminescence per well obtained by culturing with the
addition of NGF and the aptamer in mixture. The 50% inhibitory
concentration (IC50) was determined from the concentrations at two,
above and below points sandwiching the 50% inhibitory activity. The
results are shown in Table 1. IC50 value indicated as "<X" means
that the inhibitory activity was not less than 50% when the indicated
concentration X was minimum measured concentration. As a result of the
experiment, the IC50 value of the aptamers other than SEQ ID NOs:
81, 84, 86, 89 was found to be not more than 1 nM.

[0182] The 5' side of all sequences of the internal loops 1 in these
aptamers was CCU and the 3' side was UGUU. In addition, loop 2 contained
a consensus sequence shown by 5'-UUUCCXU-3'. Here, X is either G or U.
The final base pair of stem 1 was always U-a. The 1st, 8th and 9th base
pairs of stem 2 were G-C, a-U and G-C, respectively. The 2nd-7th
contained some different base pairs.

Example 12

Production of NGF Aptamer--6

[0183] To produce an aptamer that inhibits NGF but does not inhibit NT-3
and NT-4, new SELEX was performed. As a first pool, RNA pools used first
in Examples 9 and 10 were mixed at 1:1 and used. The RNA pool before
selection was mixed with NT-3 (manufactured by R&D Systems, 294 pmol),
NT-4 (manufactured by R&D Systems, 179 pmol) and BDNF (manufactured by
R&D Systems, 148 pmol), and the mixture was added to beads with NGF (380
pmol) immobilized thereon.

[0184] After the completion of 4 rounds, the sequences of 48 clones were
confirmed to find no sequence convergence. Some single sequences had the
same sequences as SEQ ID NOs: 27, 28, 34, 64, 72. Novel 14 sequences were
selected and the secondary structure was predicted using the MFOLD
program to find that all contained the consensus secondary structure 1.
Thus, these aptamers were subjected to shortening to 40 mer in the same
manner as with the consensus secondary structure 1. The binding activity
of these shortened forms to NGF and NT-3 was measured by the surface
plasmon resonance method. For the measurement method, BIAcore2000
manufactured by BIAcore was used and CM5 reactive with amino group was
used as a sensorchip, as shown in Example 1. The protein was immobilized
in the same manner as in Example 1 by using ethyl-3-carbodiimide
hydrochloride and N-hydroxysuccinimide. Human NGF or NT-3 was dissolved
in an immobilization solution (10 mM sodium acetate, pH 6) and used at
25-40 μg/ml. After protein immobilization, blocking with
ethanolamine-HCl was performed. The amount of immobilized NGF and NT-3
was 3,000-4,000 RU and 3,000-5,000 RU, respectively. The aptamer for an
analyte was prepared to 0.15 μM-0.5 μM. The running buffer and
regeneration solution were the same as those in Example 1. NT-3 was
immobilized on FC2, and NGF on FC3, and the final sensorgram was obtained
by subtracting the results of FC1. Consequently, 7 sequences were found
to strongly bind to NGF. On the other hand, almost no sequences bound to
NT-3.

[0185] In the same manner as in Example 3, the neurite outgrowth
inhibitory activity was measured to find that the aptamer shown by SEQ ID
NO: 93-98 showed a strong inhibitory activity at 10 nM. The nucleotide
sequences actually obtained, which correspond to each SEQ ID NO, are
shown below.

[0186] The 5' side of all sequences of the internal loops 1 in these
aptamers was CCU and the 3' side was UGUU. In addition, loop 2 contained
a consensus sequence shown by 5'-UUUCCXU-3'. Here, X is either G or a.
The final base pair of stem 1 was always U-a. The 2nd, 5th, 8th and 9th
base pairs of stem 2 were C-G, C-G, a-U and G-C, respectively. There were
some different base pairs in the others.

[0187] SELEX was performed up to 7 rounds and the sequences of 48 clones
were confirmed to find that most sequences converge one kind of sequence.
13 single sequences were selected from the rest, and the binding activity
to NGF and NT-3 was examined by the surface plasmon resonance method and
in the same manner as above. Consequently, 7 sequences were found to
strongly bind to NGF. On the other hand, almost no sequences bound to
NT-3.

[0188] The secondary structure of these aptamers was predicted using the
MFOLD program to find that all did not have the consensus secondary
structure 1. In the same manner as in Example 3, the neurite outgrowth
inhibitory activity was measured to find that the aptamers shown by SEQ
ID NOs: 99 and 100 showed a strong inhibitory activity at 10 nM. On the
other hand, the TF-1 cell proliferation inhibitory activity was measured
in the same manner as in Example 8 to find no inhibitory activity. The
nucleotide sequences actually obtained, which correspond to each SEQ ID
NO, are shown below.

[0189] New SELEX was performed in the same manner as in Example 12 to
produce an aptamer that inhibits NGF but does not inhibit NT-3 and NT-4.
The template and primer first used for RNA pool are as described below.

[0190] The template sequence was based on the sequence shown by SEQ ID NO:
82, stem 2 of the consensus secondary structure 1 was doped with 30% of
random sequence, and internal loop 1 and loop 2 sections were completely
randomized (n).

[0191] After the completion of 4 rounds, the sequences of 48 clones were
confirmed to find that 21 clones were identical with the sequence shown
by SEQ ID NO: 22. Of the remaining sequences, 2 clones were the same, and
others were single sequences. 13 sequences were selected therefrom and
the secondary structure was predicted using the MFOLD program to find
they had the consensus secondary structure 1. Then, the aptamers were
subjected to shortening to 40 mer in the same manner as with consensus
secondary structure 1. The binding activity of the shortened aptamers was
confirmed by the surface plasmon resonance method in the same manner as
in Example 12. NT-4 was measured in the same manner as in NT-3. As a
result, it was found that the shortened forms strongly bind to NGF. On
the other hand, the binding to NT-3 and NT-4 was weak.

[0192] In the same manner as in Example 3, the neurite outgrowth
inhibitory activity of these shortened forms was measured to find that
the aptamers shown by SEQ ID NOs: 101 and 102 showed a strong inhibitory
activity as evidenced by an IC50 value of 1 nM or below (Table 1).
On the other hand, the TF-1 cell proliferation inhibitory activity was
measured in the same manner as in Example 8 to find that the IC50
value of the same two aptamers was not less than 1 nM. The nucleotide
sequences actually obtained, which correspond to each SEQ ID NO, are
shown below.

[0193] To obtain an aptamer having consensus secondary structure 1, which
inhibits NGF but does not inhibit other neurotrophins, sequences were
analyzed using high-speed sequencer GS FLX (manufactured by Roche). While
sequence analysis of 48 clones was performed by Sanger sequencing in
Example 1, use of a high-speed sequencer enables analyses of tens of
thousands of sequences. The measurement and data analysis were performed
in Operon, and sample preparation was performed according to the protocol
of Operon. The measurement target DNA was an equimolar mixture of DNA
pools after the completion of 7, 9 and 10 rounds obtained by SELEX in
Example 8, 4 and 5 rounds obtained by SELEX in Example 12, and 3 and 4
rounds obtained by SELEX in Example 13.

[0194] The total number of the obtained sequences was 69249. Among them,
40077 sequences contained a completely identical FLX primer sequence or a
FLX primer sequence wherein one base is substituted, and a partial
sequence of N40 with a length of 40. The secondary structure of these
sequences was predicted using the RNAfold program to find 22453 sequences
containing the same structure as the consensus secondary structure 1.
When compared to the sequences obtained in Examples 10, 12, 13 by Sanger
sequencing, 99% were novel sequences. Among the novel sequences, 1615
kinds of sequences contained convergence, and 4168 sequences were single
sequences. Novel 52 sequences that emerged highly frequently were
selected therefrom, and subjected to shortening to 40 mer to afford the
shape of consensus secondary structure 1. In addition, 10 single
sequences obtained in Example 13 by Sanger sequencing were picked up
again, and subjected to shortening to 40 mer in the same manner.

[0195] The shortened sequences were measured for the binding to NGF, NT-3,
NT-4 by the surface plasmon resonance method. The measurement was
performed in the same manner as in Example 13. As a result, all sequences
bound to NGF, and particularly, the following 15 sequences showed strong
binding. On the other hand, they were scarcely bound to NT-3 and NT-4.

[0196] Therefore, the neurite outgrowth inhibitory activity of the 15
sequences was measured by a method similar to that in Example 3. As a
result, it was found that all aptamers had an IC50 value of not more
than 1 nM (Table 1). In addition, the TF-1 cell proliferation inhibitory
activity was measured by a method similar to that in Example 8 to find
that the aptamers shown by SEQ ID NOs: 111, 112, 114-117 had an IC50
value of not more than 1 nM (Table 1).

[0197] The nucleotide sequences actually obtained, which correspond to
each SEQ ID NO, are shown below. SEQ ID NO: 111 is the sequence obtained
in Example 13 by Sanger sequencing.

[0198] The 5' side of all the sequences of the internal loops 1 in these
aptamers was CCU and the 3' side was UGUU. In addition, loop 2 contained
a consensus sequence shown by 5'-UUUCCXU-3'. Here, X is either G or U.
The final base pair of stem 1 was always U-a. The 8th and 9th base pairs
of stem 2 were a-U and G-C, respectively. The 1st to 7th contained some
different base pairs.

Example 15

Modification of Shortened Aptamers

[0199] To enhance the stability of the aptamer in blood, variants wherein
the modification at the 2'-position of ribose has been replaced were
prepared.

[0200] The sequences of the modified forms are shown below. The
parentheses in the nucleotides show the 2'-position modification, F is
fluorine atom, M is o-methyl group, and L is Locked Nucleic Acid (LNA).
The upper-case letter shows RNA, the lower-case letter shows DNA, and idT
means inverted dT. The linker used for the 5' end was ssH Linker (SAFC)
or DMS (O)MT-AMINO-MODIFIER C6 (GLEN RESEARCH), and the linker used for
the 3' end was TFA Amino C-6 lcaa CPG (ChemGenes). PEG40GS2 is 2-branched
GS type having a molecular weight of 40000 (SUNBRIGHT GL2-400GS2
manufactured by NOF CORPORATION), PEG40TS2 is 2-branched TS type having a
molecular weight of 40000 (SUNBRIGHT GL2-400TS manufactured by NOF
CORPORATION), PEG40TS4 is 4-branched TS type having a molecular weight of
40000 (SUNBRIGHT GL4-400TS manufactured by NOF CORPORATION), PEG80TS2 is
2-branched TS type having a molecular weight of 80000 (SUNBRIGHT
GL2-800TS manufactured by NOF CORPORATION), and PEG80TS4 is 4-branched TS
type having a molecular weight of 80000 (SUNBRIGHT GL4-800TS manufactured
by NOF CORPORATION).

[0201] The binding activity of RNA shown by SEQ ID NO: 82(2) to NGF, NT-3,
NT-4 was measured by the surface plasmon resonance method in the same
manner as in Example 13. As a result, the RNA was found to have a binding
activity to any protein (FIG. 3). In addition, whether the aptamer shown
by SEQ ID NO: 82(2) inhibits the binding of NGF and its receptor (TrkA or
p75) was examined in the same manner as in Example 2 by the surface
plasmon resonance method. As a result, it was found that the aptamer
strongly inhibits the binding of NGF and the both receptors (FIGS. 4 and
5).

[0202] The neurite outgrowth inhibitory activity of all of the
above-mentioned modified forms was measured by a method similar to that
in Example 3. As a result, the IC50 value of all modified forms
other than SEQ ID NO: 62(1) was not more than 1 nM (Table 1).
Particularly, the IC50 value of the aptamer shown by SEQ ID NO: 162
was 0.033 nM. Moreover, the TF-1 cell proliferation inhibitory activity
was measured by a method similar to that in Example 8 to find that almost
all aptamers had an IC50 value of not more than 1 nM (Table 1).
Particularly, the IC50 value of the aptamer shown by SEQ ID NO: is
162 was 0.014 nM. The IC50 value of the aptamer shown by SEQ ID NO:
62(1) was 0.49 nM.

[0203] From the above, it was shown that the aptamers can maintain the
inhibitory activity even when the modification at the 2'-position of
ribose is changed.

Confirmation of Cross-Reactivity with Other Neurotrophins by a TF-1 Cell
Proliferation Inhibition Assay

[0204] Using TF-1 cells, whether NGF aptamer inhibits BDNF, NT-3, NT-4 was
examined. Human receptor genes (TrkB, TrkC, p75) for respective
neurotrophic factors were introduced into TF-1 cells (ATCC Number:
CRL-2003), which is a human erythroleukemic cell line, by using a
retrovirus vector to give cells that highly express these receptors
stably. TF-1 cells introduced with TrkB and p75 were used for the
evaluation of inhibitory activity against BDNF, TF-1 cells introduced
with TrkC and p75 were used for the evaluation against NT-3, and TF-1
cells introduced with TrkB alone were used for the evaluation against
NT-4. These cells were suspended in an RPMI-1640 medium containing 20%
fetal bovine serum, and seeded in a white 96 well flat-bottom plate at
1000 cells (50 μL) per well. Thereto was added a mixed solution 50
μL of human BDNF (final concentration 0.074 nM) or NT-3 (final
concentration 0.074 nM) or NT-4 (final concentration 0.071 nM) and the
aptamer (final concentration 1 μM-0.01 nM), which had been pre-reacted
at room temperature for 30 min in a serum-free RPMI-1640 medium, 3 days
later, 100 μL of CellTiter-Glo reagent for CellTiter-Glo Luminescent
Cell Viability Assay (manufactured by Promega) was added to each well,
chemiluminescence was measured by a microplate reader. With the amount of
luminescence per well obtained by the addition of BDNF or NT-3 or NT-4
alone and culture of the cells for 3 days as inhibitory activity 0%, and
that of the well obtained by culture for 3 days without addition of BDNF
or NT-3 or NT-4 as inhibitory activity 100%, the inhibitory activity of
the aptamer was calculated from the amount of luminescence per well
obtained by culturing with the addition of BDNF or NT-3 or NT-4 and the
aptamer in mixture. When the inhibitory activity was 0 or below, `0%` is
indicated. The 50% inhibitory concentration (IC50) was determined
from the concentrations at two, above and below points sandwiching the
50% inhibitory activity. The experiment results are shown in Table 2. An
IC50 value indicated as ">X" means that the inhibitory activity
was not more than 50% when the indicated concentration X was the maximum
measured concentration. N.D. means not measured.

[0205] All the tested aptamers showed a strong inhibitory activity. The
IC50 values thereof are partly shown in Table 2. As the inhibitory
activity of the aptamers of the present invention described in Table 2,
the IC50 value to NGF was not more than 0.1 nM, whereas that to BDNF
was not less than 1000 nM. The IC50 value to NT-3 varied from 0.97
nM to not less than 10 nM depending on the aptamer. The IC50 value
to NT-4 varied from not more than 3 nM to not less than 30 nM depending
on the aptamer.

[0206] To study the analgesic action of NGF aptamer on NGF-induced pain, a
thermal hyperalgesia model induced by subcutaneous administration of NGF
to rat hind paw was used. For the experiment, Jcl:SD rats (6-week-old)
were used. As an index of thermal hyperalgesia, response latency of
escape behavior to infrared irradiation from a plantar heat stimulation
measuring apparatus (manufactured by Ugo Basile) to the planta was used.
On the previous day of the test, acclimation to the evaluation system was
performed. Before administration on the day of the test, escape response
latency was measured, and animals that showed not less than 10 sec and
less than 20 sec were used. Human β-NGF (R&D Systems, final
concentration 50 μg/ml) and a test substance were mixed with vehicle
(20 mM Tris-HCl (pH 7.6), 145 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 1.8
mM CaCl2, 0.1% BSA), incubated at room temperature for 30 min, and
subcutaneously administered to the left hind sole at 10 μl. The escape
response latency was measured 5 hr later. The aptamer represented by SEQ
ID NO: 153 was administered at a final concentration of 50 mg/ml (molar
ratio relative to NGF:1000-fold). As a control, vehicle or a mixture of
vehicle and NGF was administered in the same manner. The results are
shown in Table 3 (Mean±SEM, n=9).

[0207] At 5 hr after administration, the NGF group showed significantly
low escape response latency as compared to the vehicle group (p<0.01).
At 5 hr after the administration, the escape response latency of the
aptamer administration group was high (p<0.01) as compared to the NGF
alone administration group. From the above results, it was found that
this aptamer can be used as a drug for NGF-induced pain.

[0208] To study the efficacy of NGF aptamer therapy, a postoperative pain
model which was to have induced thermal hyperalgesia was used. For the
experiment, Crl:CD(SD) rats (5-week-old) were used. The tip of a catheter
was indwelled in the femoral vein, the other tip was exposed from the
back of the rat. One week later, Quick connect infusion system
(manufactured by Strategic applications incorporated) was set on the rat,
thermal hyperalgesia was evaluated one week later. As an index of thermal
hyperalgesia, response latency of escape behavior to infrared irradiation
from a plantar heat stimulation measuring apparatus (manufactured by Ugo
Basile) to the planta was used. Acclimation to the evaluation system was
performed 3 days before the start of the test. On the day of the test,
escape response latency was measured, and animals that showed not less
than 10 sec and less than 20 sec were used. The NGF aptamer dissolved in
saline intravenously administered with a syringe pump (manufactured by
TERUMO CORPORATION) in a sustained manner. As the NGF aptamer, the
aptamer shown by SEQ ID NO: 82(56) (administered at 21.2 mg/240 ml/kg/96
hr) and the aptamer shown by SEQ ID NO: 82(55) (administered at 10.08
mg/240 ml/kg/96 hr) were used. As a control, vehicle was administered in
the same manner. At 1 hr from the start of the administration, the skin
and fascia of the right hind sole were incised, the flexor was vertically
bisected, and the skin was sutured. The escape response latency was
measured after incision operation, and 1, 2, 3, 4 days thereafter. The
results are shown in Table 4.

[0209] The vehicle group showed significantly low (p<0.01) escape
response latency at 1, 2, 3, 4 days after administration-incision
operation as compared to before administration-incision operation. At 1,
2, 3, 4 days after administration-incision operation, the escape response
latency of any aptamer administration group was significantly high
(p<0.01) as compared to the vehicle group. The results of the
experiment are shown in Table 4 and Table 5 (Mean±SEM, n=8-9). This
has revealed that an anti-NGF aptamer has an analgesic action on
postoperative pain model.

[0210] The aptamer of the present invention can be useful as medicaments,
diagnostic agents or reagents for diseases such as algia, inflammatory
disease and the like. The aptamer and the complex of the present
invention can also be useful for the purification and concentration of
NGF, as well as detection and quantification of NGF.

[0211] This application is based on a patent application No. 2011-213585
filed in Japan (filing date: Sep. 28, 2011), the contents of which are
incorporated in full herein.